Enzymes are proteins having catalytic properties. A "catalyst" increases the rate of a particular chemical reaction without itself being consumed or permanently altered; at the end of a catalyzed reaction, the main reaction products have undergone transformation into new products, but the catalyst appears unchanged in form and quantity. Thus the presence of a small number of enzyme molecules in a reaction mixture involving a substrate can convert a greater number of the substrate molecules to products. Similarly, an increase in the amount of an enzyme in a sample, such as, for example, a clinical sample (e.g., serum, plasma, cerebro spinal fluid, urine), can be detected with great specificity because of the unique and characteristic effect that the enzyme has on the chemical reaction which it catalyzes.
Isoenzymes are two or more enzymes which catalyze the same (or similar) specific reactions but which have different physical properties, e.g., electrophoretic mobility, resistance to chemical or thermal inactivation, etc. Thus, while in a class of isoenzymes, each will have the same catalytic function, subtle, yet detectable differences between each can be determined such that, in a sample material, the relative distribution of each isoenzyme having the same catalytic function can be determined.
Analysis of the presence of enzymes from clinical samples is diagnostically valuable. Specific enzymes are associated with specific tissue sources. For example, the enzyme lactate dehydrogenase ("LDH") is found principally in the heart, liver, skeletal muscle and lymph nodes. Creatine kinase ("CK") is found principally in skeletal muscle, brain, heart and smooth muscle. Cholinesterase ("CHE") is found principally in the liver. When these tissue sources are damaged (due to internal causes, such as disease, or external sources, such as alcohol), there is typically a release of the enzyme(s) associated therewith into the blood stream. Accordingly, a clinical sample can be analyzed and if elevated levels of, for example, LDH, are identified, possible damage to the associated tissues is evident.
LDH has five isoenzymes. It is well known that certain diseases will cause a change in the relative distribution of LDH isoenzymes occurring in serum. LDH is a hydrogen transfer enzyme that catalyzes the oxidation of L-lactate to pyruvate with the mediation of nicotinamide adenine dinucleotide (NAD.sup.+) as the hydrogen acceptor. Thus, in the presence of LDH, L-lactate and NAD.sup.+ will be catalytically converted to pyruvate and NADH. NADH production can be measured and the amount can be correlated with the amount of LDH in the sample. Each LDH isoenzyme has a unique concentration in a given sample. Similarly each isoenzyme is capable of catalyzing the L-Lactate/NAD.sup.+ reaction. Thus each isoenzyme will produce different amounts of NADH by such catalysis.
Electrophoretic separation on agarose gels or cellulose acetate is a well known procedure used to demonstrate the presence of LDH isoenzymes in a sample. Typically, a clinical sample (e.g. serum) is inserted into a well in the gel surface and a voltage impressed across the gel. Since each isoenzyme has a unique electrophoretic mobility, the voltage separates the isoenzymes from each other. A reaction mixture is then layered over the separation medium and, following sufficient incubation, the NADH generated over the individual LDH zones is detected (typically by fluorescence when the NADH is excited by ultraviolet light). Thereafter, the gel patterns may be read directly by observing the relative intensities of the bands, or scanned by, e.g., color densitometer instruments, such that relative peak distribution can be obtained. Such an electrophoresis system is commercially available from Beckman Instruments, Inc. (Fullerton, Calif., USA) under the trademarks APPRAISE.RTM. and PARAGON.RTM..
Many clinically significant isoenzyme product patterns have been correlated with certain disease states. Again focusing on LDH, a normal LDH distribution pattern will evidence a "darker" band (or higher peak) for LDH-2 vis-a-vis LDH-1, whereas a serum sample obtained from an individual having acute myocardial infarction will evidence a darker (or higher) LDH-1 band vis-a-vis LDH-2, the so-called "flipped" LDH-1.
As noted, isoenzymes have different physical properties, e.g., different electrophoretic mobilities. Accordingly, isoenzymes lend themselves to analysis by capillary zone electrophoresis ("CZE"). CZE is a technique which permits rapid and efficient separations of charged substances. In general, CZE involves introduction of a sample into a capillary tube, i.e., a tube having an internal diameter from about 5 to about 2000 microns, and the application of an electric field to the tube. The electric potential of the field both pulls the sample through the tube and separates it into its constituent parts. Each constituent of the sample has its own individual electrophoretic mobility; those having greater mobility travel through the capillary tube faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones.
CZE can be generally separated into two categories based upon the contents of the capillary columns. In "gel" CZE, the capillary tube is filled with a suitable gel, e.g., polyacrylamide gel. Separation of the constituents in the sample is predicated in part by the size and charge of the constituents travelling through the gel matrix. In "open" CZE, the capillary tube is filled with an electrically conductive buffer solution. Upon ionization of the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution (the buffer solution and the sample being analyzed), must also flow in this direction to maintain electroneutrality. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species regardless of charge, towards the cathode. Fused silica is principally utilized as the material for the capillary tube because it can withstand the relatively high voltage used in CZE, and because the inner walls of a fused silica capillary ionize to create the negative charge which causes the desired electroendosomatic flow.
The inner wall of the capillaries used in CZE can be either coated or uncoated. The coatings used are varied and known to those in the art. Generally, such coatings are utilized in order to reduce adsorption of the charged constituent species to the charged inner wall. Similarly, uncoated columns can be used. In order to prevent such adsorption, the pH of the running buffer, or the components within the buffer, are manipulated.
While the different electrophoretic mobilities of isoenzymes suggests the applicability of CZE analytical techniques, a practical problem exists as to the parameters of such testing. This is because in order to determine the presence of isoenzymes in a sample, it is necessary to rely upon their catalytic activity relative to a substrate. Thus, in the electrophoresis system described above, separation of the isoenzymes must take place before addition of the substrate. However, this is not possible with CZE because there is presently no practical way to "add" the substrate to the sample after the constituent parts thereof have been separated.
What is needed, then, is a method for analyzing isoenzymes that exploits the speed and accuracy of CZE techniques.